Having reached the milestone of sequencing entire genomes, fundamental questions in understanding human biology are now: How are genomes organized in living cells and what are the mechanisms that determine what gene expression programs are active. Our cell biological studies of genomes and the cell nucleus are aimed at uncovering fundamental concepts of genome organization and nuclear function in vivo and they are providing opportunities for applying these principles to human disease diagnosis, therapeutics and bioengineering. To understand the nuclear environment in which genomes are expressed, we are probing the biophysical properties of proteins and chromatin using in vivo imaging. To this end, we generate functional, fluorescently labeled molecules, which can be introduced into cells and visualized in the nucleus of living cells by time-lapse microscopy. Using these methods we have succeeded in analyzing gene expression processes in living cells to study the structure-function relationship of the mammalian cell nucleus. Recently we have initiated studies to measure biophysical properties of proteins in vivo by using photobleaching techniques in conjunction with kinetic modeling and computer simulation approaches. These methods provide powerful tools to analyze for the first time at the molecular level the action of proteins in living cells and in real time. The second major issue in understanding how genomes function in cells is to determining how genomes are organized. To this end we have initiated studies to map the spatial organization of chromosomes within the nucleus and we are testing whether positioning of chromosomes affects gene expression and regulation. These studies must be considered first steps towards rigorous interphase cytogenetic methods. Finally, we are using our cell biological approaches to investigate the cellular organization of alternative splicing by investigating the differential association of pre-mRNA splicing factors with alternatively spliced transcripts. We have also initiated a study to apply our knowledge of alternative splice site selection to correct aberrant splicing in human disease genes with the hope to restore normal splicing in a 'RNA-therapy' approach.